Modulation of immune system with Δ5-androstenes

ABSTRACT

Alzheimer&#39;s disease and immune deficiency disorders may be effectively treated by administering a Δ5-Androstene-3β-ol-17-one having a C 7  substituent selected from the group consisting of oxo, hydroxy and groups convertible thereto by hydrolysis.by administering a therapeutic amount of a Δ5-Androstene-3β-ol-17-one having a C 7  substituent selected from the group consisting of oxo, hydroxy and groups convertible thereto by hydrolysis.

This is a continuation-in-part of U.S. patent application Ser. No.867,288 filed 10 Apr. 1992, pending which is a continuation of U.S.patent application Ser. No. 575,156 filed 29 Aug. 1990, now abandoned.

FIELD OF THE INVENTION

Broadly, the invention relates to the use of steroids for effecting adesired biological response. Specifically, the invention relates to theuse of Δ5-androstenes for retarding the degenerative effects ofAlzheimer's disease and modulating the antibody responsiveness of theimmune system.

BACKGROUND Alzheimer's Disease

Alzheimer's disease is a degenerative brain disease characterized by theloss of nerve cells in the cerebral cortex. The disease is the leadingcause of presenile dementia. Among the deleterious effects are speechdisturbances, severe short term memory loss and disorientation. Thedisease results in a progressive loss of the mental facilities.

Despite years of extensive research, investigators have yet tounderstand the cause of the disease and have to date been unable to findan effective treatment. However, it is generally thought that thedisease is associated with a deficiency of the neurotransmitteracetylcholine.

Accordingly, a substantial need exists for a therapeutic agent effectivefor retarding the deleterious effects of Alzheimer's disease.

Immune Response

The immune system protects against the introduction and advancement ofpathogenic microorganisms through activation of T and B lymphocytes abdmacrophages. Upon detection of an antigen, such as a pathogenicmicroorganism, T cells are activated to produce lymphokines thatinfluence the activities of other host cells and the B cells mature toproduce immunoglobulins or antibodies that react with the antigen.

Immune senescence results in a decrease in the antibody responsivenessof the immune system and thereby retards the ability of the system toimmunize the body against pathogenic microorganisms. Such a depressedimmune system results in an increase in the frequency and severity ofpathogenically induced maladies and possibly death.

Immune senescence may result as a natural consequence of aging or as adeleterious effect of pathological microorganism(s). Immune senescenceis one of the major health problems of our time with a general consensusand within the medical profession that the problem may soon reachepidemic proportions.

Accordingly, a substantial need exists for a therapeutic agent effectivefor inducing an immunomodulatory response to immune senescence.

SUMMARY OF THE INVENTION Alzheimer's Disease

Alzheimer's disease may be treated by administering a therapeutic amountof a Δ5-Androstene-3β-ol-17one having a C₇ substituent selected from thegroup consisting of oxo, hydroxy and groups convertible thereto byhydrolysis. Such treatment is effective for retarding the degenerativeeffects of Alzheimer's disease, including specifically, but notexclusively, deterioration of the central nervous system, loss of metalfacilities, loss of short term memory, and disorientation.

These steroids are particularly effective for use in treatingAlzheimer's disease as they, contrary to other Δ5-Androstenes, providethe desired biological response without stimulating the undesiredproduction of additional sex hormones.

Immune Response

Immune senescence may be modulated by administering a therapeutic amountof a Δ5-Androstene-3β-ol-17-one having a C₇ substituent selected fromthe group consisting of oxo, hydroxy and groups convertible thereto byhydrolysis.

These steroids are particularly effective for modulating the immunesystem as they, contrary to other Δ5-Androstenes, provide the desiredbiological response without stimulating the undesired production ofadditional sex hormones.

DETAILED DESCRIPTION OF THE INVENTION INCLUDING A BEST MODE

Alzheimer's disease and immune deficiency disorders may be effectivelytreated by administering a Δ5-Androstene-3β-ol-17-one having a C₇substituent selected from the group consisting of oxo, hydroxy andgroups convertible thereto by hydrolysis.

These steroids may also be administered as a carbamate, enanthate orother such derivative capable of releasing the specified steroid withinthe intestinal tract, blood and/or body tissues.

Synthesis (1) Δ5-Androstene 3β,7-diol, 17-one (7-hydroxy DHEA)

Δ5-Androstene 3β,7α-diol, 17-one (7-hydroxy DHEA) can be synthesizedfrom commercially available DHEA acetate (10) by sequentiallysynthesizing:

Δ5-Androstene-3β hydroxy-17-one acetate

Δ5-Androstene-3β-hydroxy-7-bromo-17-one

Δ5-Androstene-3β,7α-hydroxy-17one diacetate

Δ5-Androstene-3β,17α-hydroxy-17-one

Δ5-Androstene 3β-hydroxy-7-bromo-17-one (7-bromo DHEA) can besynthesized from Δ5-Androstene 3β-hydroxy-17-one acetate (DHEA acetate)by reacting the DHEA acetate with a brominating agent such asDibromantin (1,3 dibromo 5,5 dimethylhydantoin or N-bromo succinimide.The 7-bromo DHEA is unstable and must be used immediately in the nextstep of the process.

The 7-bromo DHEA containing an isomeric mixture of 7α-bromo DHEA and7β-bromo DHEA may be equilibrated to 7α-bromo DHEA in accordance withthe method described for a cholesterol derivative in Confalone, P.N.,Kulesha, I.D., and Uskokovic, M.R. Jour. Org. Chem., vol. 46, pp1030-1032 (198). Briefly the racemic mixture of 7-bromo DHEA iscontacted with cold anhydrous LiBr and shielded from light until thestereospecific composition is achieved.

Δ5-androstene 3β, 7-hydroxy-17-one diacetate (7-hydroxy DHEA diacetate)may be synthesized from the 7-bromo DHEA by reacting the 7-bromo DHEAwith a mixture of glacial acetic acid and powdered silver acetate atroom temperature in a suitable solvent such as methylene chloride oracetone.

Δ5-androstene 3β, 7α-hydroxy-17-one (7-hydroxy DHEA) 2 may besynthesized from the 7-hydroxy DHEA diacetate by reacting the 7-hydroxyDHEA diacetate dissolved in methanol with an aqueous solution containinga suitable base such as Na₂ CO₃.

The synthesized 7-hydroxy DHEA may then be purified by (i) evaporatingthe methanol in vacuo, (ii) extracting the 7-hydroxy DHEA into anappropriate organic solvent such as dichloromethane, (iii) evaporatingthe organic solvent in vacuo, (iv) azeotropically drying the extractedsolids containing the 7-hydroxy DHEA with a suitable organic solventsuch as ethanol, (v) dissolving the extracted solids in acetone, andthen (vi) adding a suitable precipitating agent, such as hexane, to theacetone solution to produce purified crystals of Δ5-Androstene3β,7α-diol, 17-one (7-hydroxy DHEA).

A second crop of Δ5-Androstene-3β,7α-diol-17-one (7-hydroxy DHEA)crystals may be obtained by cooling the resultant solution below roomtemperature.

(2) Δ5-Androstene-3βol 7,17-dione (7-keto DHEA)

Δ5-Androstene 3β-ol-7,17-dione can be synthesized from commerciallyavailable DHEA acetate by sequentially synthesizing:

3β-acetoxy-Δ5-androstene-17-one

3β-acetoxy-Δ5-androstene-7,17-dione

Δ5-androstene 3β-hydroxy-7,17-dione

3β-acetoxy-Δ5-androstene-7,17-dione (7-oxo DHEA acetate) can besynthesized from 3β-acetoxy-Δ5-androstene-17-one (DHEA acetate) byreacting the DHEA acetate with the oxidizing agent CrO₃ in accordancewith the procedure outlined in Fieser, L.F., Jour. Am. Chem. Soc., vol.75, pp 4386-4394 (1953).

Δ5-androstene 3β-hydroxy-7,17-dione (7-one DHEA) can be synthesized fromthe 7-one DHEA acetate and purified by employing the deesterificationand purification steps set forth above with respect to the synthesis andpurification of 7-hydroxy DHEA from 7-hydroxy DHEA diacetate.

Treatment

A subject may be treated with the steroids specified herein by any ofthe commonly accepted practices including ingestion or injection. It isbelieved that treatment at a dosage rate of about 0.1 to 2 grams ofsteroid per 100 kilograms of body weight per day, preferably 0.5 to 2grams of steroid per 100 kilograms of bodyweight per day, is generallyeffective for triggering the desired biological responses. A dose rateof less than about 0.1 grams per 100 kilograms bodyweight is generallybelieved to be insufficient to trigger the desired biological responsewhile a dose rate of greater than about 2 grams per 100 kilogramsbodyweight is believed to result in an increase in the cost of thetreatment without providing a corresponding benefit in performance. Theoptimum dose rate to be administered to a subject is case specific asthe optimum dose rate depends upon several factors including currentbody composition (percent fat), age, and the like.

Without intending to be limited thereby, we believe that the steroidsspecified herein are metabolic intermediates along the pathway toconversion of DHEA to an ultimate metabolite(s) which is responsible fortreatment of Alzheimer's disease.

A subject may be treated with one of the steroids specified herein onsubstantially any desired schedule. However, it is believed that thesteroids themselves are not stored within the body and are substantiallyremoved and/or deactivated within hours after administration.Accordingly, for optimum effectiveness the subject under treatmentshould be treated at least about every day. For reasons of conveniencethe subject under treatment may be treated less frequently, such asevery other day or once a week, when less than maximum performance isacceptable.

As is apparent from the factors which affect dosage and dose rate, eachparticular subject should be carefully and frequently reviewed and thedosage and/or dose rate altered in accordance with the particularsituation.

EXPERIMENTAL EXAMPLE I Synthesis Δ5-Androstene 3β, 7α-diol-17-one(7-hydroxy DHEA)

(Step 1) Into a two liter, triple neck, round bottom flask equipped witha magnetic stirrer and a reflux condenser was placed 1000 ml hexane (b.p69-71°), 10 grams (0.03 moles) DHEA acetate and 13.6 grams (0.16moles)NaHCO₃ to form a first mixture. The first mixture was placed under a N₂atmosphere and heated under constant agitation to reflux. Into therefluxing first mixture was added 6.11 grams (0.02 moles) Dibromantin(1,3 dibromo 5,5 dimethylhydantion) as a brominating agent to form asecond solution. The second solution gradually turned orange after whichit rapidly turned a pale white/yellow. The second solution was refluxedfor 30 minutes, cooled to room temperature and filtered through asintered glass funnel. The residue was rinsed with 50 ml dichloromethaneand the combined filtrate rotovapped to dryness at a temperature of lessthan 35° C. The dry filtrate (Δ5-Androstene 3β-ol-7-bromo-17-one) isunstable to storage and was used immediately in step two.

(Step 2) The dry filtrate was resolubilized in 80 ml of dichloromethanein a one liter stoppered flask equipped with a magnetic stirrer andplaced in an ice bath. Into the resolubilized filtrate was added 8 gramsanhydrous LiBr in 320 ml ice-cold acetone to form a third solution. Thethird solution was shielded from light and stirred continuously forthree hours. The resulting solution containing predominantly(Δ5-Androstene 3β-ol-7α-bromo-17-one) was allowed to warm briefly andused immediately in step three.

(Step 3) Into a 500 ml flask equipped with a magnetic stirrer was placed320 ml dichloromethane, 80 ml glacial acetic acid, and 26 grams ofsilver acetate to form a first suspension. The first suspension wasstirred continuously for 20 minutes at room temperature. The stirredfirst suspension was added under constant agitation into the solution ofpredominantly Δ5-Androstene 3β-ol-7α-bromo-17-one to form a secondsuspension. The second suspension was constantly stirred for 30 minutesat room temperature after which the suspension was filtered through asintered glass funnel to produce separate a solid fraction. The filteredsolid fraction was rinsed with 100 ml dichloromethane. The filtrate wasextracted three times with 1000 ml of water, remaining acetic acid wasneutralized with 5% NaHCO₃ solution, and the dichloromethane solutionwas extracted twice more with water. The organic solution containingΔ5-Androstene 3β-17α-diol-17-one diacetate was then rotovapped todryness.

(Step 4) The dried extracted solids were resolubilized in 500 mlmethanol in a one liter, triple necked flask equipped with a magneticstirrer and a reflux condenser to form a fourth solution. The fourthsolution was placed under a N₂ atmosphere and heated under constantstirring to reflux. Into the fourth solution was added 250 ml of a 5%aqueous solution of Na₂ CO₃ to form a fifth solution. The fifth solutionwas refluxed under constant agitation for 45 minutes. The methanol wasrotovapped off and the aqueous fifth solution carefully brought to a pHof 7 with an appropriate amount of glacial acetic acid. The neutralizedfifth solution was extracted twice with 100 ml of dichloromethane. Thedichloromethane solution of Δ5-Androstene 3β, 7α-diol-17-one wasrotovapped to near dryness, azeotropically dried with absolute ethanol,and then azeotropically dried twice with acetone. Warm acetone was addedto the dried extracted solids until the solids were completely dissolvedto form a sixth solution. Hexane was added to the sixth solution untilthe solution began to cloud at which time crystals of α5-Androstene3β-7α-diol-17-one began to form at room temperature.

A second crop of α5-Androstene 3β-7α-diol-17 -one crystals was obtainedby cooling the remaining sixth solution.

The product melts at about 187-189° C. and when recrystallized fromacetone/hexane melts at about 192-193° C.

EXAMPLE II Synthesis Δ5-Androstene 3β-7(αβ)-diol-17-one 7αβ-hydroxy DHEA

Δ5-Androstene 3β-7α-diol-17-one was manufactured in accordance with theprocedure set forth in Example I except that Step 2 was eliminated withthe dried filtrate from Step I simply resolubilized in the 80 ml ofdichloromethane in preparation for Step 3.

EXAMPLE III Synthesis Δ5-Androstene 3β-ol7-7,17-dione 7αβ-keto DHEA

(Step 1) Into a 50 ml flask equipped with a magnetic stirrer and a waterbath was placed 6.5 ml acetic anhydride, 23 ml acetic acid, 17 gramssodium acetate, and 2 grams DHEA acetate to form a first mixture. Intothe first mixture was added 2 grams chromium trioxide over a thirtyminute period to form a second mixture. The first mixture was maintainedat a constant temperature of 56-58° C. and continuously agitated duringaddition of the chromium trioxide. The second mixture was maintained at56-58° C. and continuously agitated for an additional hour after whichthe second mixture was cooled and slowly poured under continuousagitation into 600 ml of ice water to form a precipitate. The flocculentprecipitate was collected on a sintered glass funnel and washed withwater until no longer green. After drying in vacuo over P₂ O₅ theproduct was dissolved in methanol and recrystallized to yieldsubstantially pure Δ5-Androstene 3β-acetoxy-7,17-dione having a meltingpoint of about 191-192° C.

(Step 2) The precipitate was resolubilized in 500 ml of methanol in aone liter, triple necked, round bottom flask equipped with a magneticstirrer and reflux condenser to form a third solution. The thirdsolution was placed under a N₂ atmosphere and heated under constantagitation to reflux. Into the third solution was added 250 ml of a 5%solution of Na₂ CO₃ to form a fourth solution. The fourth solution wasrefluxed under constant agitation for 45 minutes. The methanol wasrotovapped off and the aqueous fourth solution carefully brought to a pHof 7 with an appropriate amount of glacial acetic acid. The neutralizedfourth solution was extracted with two 100 ml portions ofdichloromethane, and two portions combined, and the dichloromethaneevaporated in vacuo. The extracted solids were then azeotropically driedfirst with absolute ethanol and then with two separate portions ofacetone. Methanol was added to the dried extracted solids until thesolids were completely dissolved to form a fifth solution. Hexane wasadded to the fifth solution until the solution began to cloud at whichtime crystals of Δ5-Androstene 3β-ol-7,17-dione began to form at roomtemperature.

A second crop of Δ5-Androstene 3β-ol-7,17-dione crystals was obtained bycooling the remaining sixth solution.

The resultant product had a melting point of about 235-238° C.

EXAMPLE IV Immunomodulatory Effect of Δ5-Androstene 3β-ol-7,17-dione(7-Oxo DHEA)

Thirty (30) acclimated and Thoren-unit housed one month old Balb/c micewere separated into six (6) groups of five and bled retro-orbitallyunder Metofane® anesthesia to obtain the pre-vaccination serum.Treatments and doses for all groups are set forth in Table 1 below. TheDHEA utilized in the trials was obtained from Steraloids, Inc. The 7-OxoDHEA was synthesized by the procedure of Example III.

                  TABLE ONE                                                       ______________________________________                                                Treatment     Steroid   Treatment                                     Group   Composition   Dose/#    Site                                          ______________________________________                                        A       7-Oxo-DHEA    500    μg/2                                                                            Contralateral                               B       DHEA          500    μg/2                                                                            Contralateral                               C       7-Oxo-DHEA    5      μg                                                                              Ipsilateral                                 D       Olive Oil     0           Ipsilateral                                 E       DHEA          50     μg                                                                              Ipsilateral                                 F       7-Oxo-DHEA    50     μg                                                                              Ipsilateral                                 ______________________________________                                    

Mice receiving two treatments (2) received the first treatment threedays before vaccination and the second treatment at the time ofvaccination. All other treatments were administered at the time ofvaccination. The vaccine consisted of 0.17 ml trivalent influenzavaccine (A/Taiwan/H3N2/868, A/Panama/H1N1/91, B/Beijing). Mice were bledthree weeks after vaccination to obtain post-vaccination serum.

Determination of the concentration of influenza antibodies in thepre-vaccination and post-vaccination sera were determined by ELISA atdilutions of 1:1000, 1:4000 and 1:16000 with antibody levels reported inoptical density at 405 nm with increased optical density indicatingincreased antibody concentration.

The post vaccination optical density for each group is depicted bydilution in Tables Two (1:1000), Three (1:4000) and Four (1:16000). Thebaseline optical densities for pre-vaccination sera ranged from valuesof 0.00 to 0.25 with an average of 0.08. The background optical densityaveraged 0.1.

                  TABLE TWO                                                       ______________________________________                                        Optical Density                                                               Post-Vaccination serum                                                        1:1000 Dilution                                                                        Density      Density  Density                                        Group    (Beijing)    (Taiwan) (Panama)                                       ______________________________________                                        A        2.26         1.64     1.85                                           B        1.25         1.59     1.76                                           C        1.35         1.89     1.37                                           D        0.87         1.18     1.01                                           E        1.71         1.44     1.17                                           F        2.06         1.19     0.93                                           ______________________________________                                    

                  TABLE THREE                                                     ______________________________________                                        Optical Density                                                               Post-Vaccination serum                                                        1:4000 Dilution                                                               Group   Beijing       Taiwan  Panama                                          ______________________________________                                        A       1.92          0.82    1.20                                            B       0.93          0.90    1.16                                            C       0.85          1.43    0.64                                            D       0.47          0.84    0.39                                            E       1.73          0.71    0.63                                            F       2.25          0.61    0.35                                            ______________________________________                                    

                  TABLE FOUR                                                      ______________________________________                                        Optical Density                                                               Post-Vaccination serum                                                        1:16000 Dilution                                                              Group   Beijing       Taiwan  Panama                                          ______________________________________                                        A       0.95          0.32    0.48                                            B       0.52          0.41    0.38                                            C       0.38          0.35    0.27                                            D       0.15          0.22    0.14                                            E       0.71          0.28    0.24                                            F       0.99          0.23    0.09                                            ______________________________________                                    

A Bonferroni/Dunn analysis was conducted upon the test results tocompare post vaccination antibody responses to combined antigens. Theresults of this analysis are set forth in Table Two BD (1:1000), threeBD (1:4000) and Four BD (1:16000).

                  TABLE TWO BD                                                    ______________________________________                                        Bonferroni/Dunn Analysis                                                      1:1000 Dilution                                                               Group   Mean Diff.    Crit. Diff.                                                                             P-Value                                       ______________________________________                                        A B     .483          .953      .1262                                         A C     .267          .953      .3946                                         A D     .707          1.004     .0355                                         A E     .238          1.004     .4709                                         A F     .180          1.004     .5857                                         B C     -.216         .898      .4650                                         B D     .223          .953      .4760                                         B E     -.245         .953      .4351                                         B F     -.303         .953      .3344                                         C D     .439          .953      .1635                                         C E     -.029         .953      .9266                                         C F     -.087         .953      .7806                                         D E     -.468         1.004     .1591                                         D F     -.527         1.004     .1141                                         E F     -.058         1.004     .8596                                         ______________________________________                                    

                  TABLE THREE BD                                                  ______________________________________                                        Bonferroni/Dunn Analysis                                                      1:4000 Dilution                                                               Group   Mean Diff.    Crit. Diff.                                                                             P-Value                                       ______________________________________                                        A B     .493          .989      .1326                                         A C     .219          .989      .5004                                         A D     .597          1.043     .0853                                         A E     .092          1.043     .7871                                         A F     -.077         1.043     .8229                                         B C     -.274         .933      .3726                                         B D     .103          .989      .7505                                         B E     -.401         .989      .2202                                         B F     -.570         .989      .0832                                         C D     .377          .989      .2481                                         C E     -.127         .989      .6964                                         C F     -.296         .989      .3639                                         D E     -.504         1.043     .1446                                         D F     -.673         1.043     .0529                                         E F     -.169         1.043     .6217                                         ______________________________________                                    

                  TABLE FOUR BD                                                   ______________________________________                                        Bonferroni/Dunn Analysis                                                      1:16000 Dilution                                                              Group   Mean Diff.    Crit. Diff.                                                                             P-Value                                       ______________________________________                                        A B     .132          .456      .3810                                         A C     .178          .456      .2384                                         A D     .322          .481      .0452                                         A E     .063          .481      .6886                                         A F     -.010         .481      .9495                                         B C     .046          .430      .7448                                         B D     .190          .456      .2077                                         B E     -.068         .456      .6486                                         B F     -.142         .456      .3461                                         C D     .144          .456      .3383                                         C E     .114          .456      .4465                                         C F     .188          .456      .2133                                         D E     .258          .481      .1057                                         D F     .332          .481      .0391                                         E F     .073          .481      .6427                                         ______________________________________                                    

CONCLUSIONS

The DHEA and Δ5-Androstene-3β-ol-7,17-dione (7-Oxo DHEA) did not induceclinically apparent toxicity.

The immune response to Taiwan A/H1N1 varied the least between treatmentgroups. Healthy mice normally respond well to this virus. Accordingly,it appears that treatment with Δ5-Androstene-3β-ol-7,17-dione does notenhance the immune response when the normal immune response is normally"optimally immunogenic".

Δ5-Androstene-3β-ol-7,17-dione contralateral site injection produced thegreatest response to the A/Panama/H1N1/91 and B/Beijing viruses. Healthymice normally do not respond well to this virus. Accordingly, it appearsthat treatment with Δ5-Androstene-3β-ol-7,17-dione does enhance theimmune response when the normal immune response is less than optimal.

What is claimed is:
 1. A method for modulating the immune system of amammal comprising the step of administering to the mammal an effectiveamount of a steroid selected from the group consisting ofΔ5-Androstene-3β-ol-17-one having a C₇ substituent selected from thegroup consisting of oxo, hydroxy and groups convertible thereto byhydrolysis.
 2. The method of claim 1 wherein the step of administeringthe steroid to a mammal comprises the step of administering the steroidto a human.
 3. The treatment method of claim 1, wherein the step ofadministering the steroid comprises the step of injecting the steroid.4. The treatment method of claim 1 wherein the step of administering thesteroid comprises the step of inducing ingestion of the steroid.